CN105420182A - Serum-free medium for umbilical cord mesenchymal stem cells - Google Patents
Serum-free medium for umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention provides a serum-free medium for umbilical cord mesenchymal stem cells and belongs to the technical field of cell culture. The serum-free medium for umbilical cord mesenchymal stem cells comprises a basal culture medium body and added ingredients, wherein the basal culture medium body is a DMEM culture medium; the added ingredients include a basic fibroblast growth factor hFGF, a epidermal growth factor hEGF, insulin hI, a leukaemia inhibitory factor hLIF and astragalus polysaccharide. The umbilical cord mesenchymal stem cells are subjected to subculture and amplification in the medium, and the surfaces of the cultured cells are marked and analyzed. The serum-free medium for umbilical cord mesenchymal stem cells overcomes the defect of exogenous pollution of serum and solves the problem of contradiction between the cell expansion number and cost reduction. The medium is free of serum, thereby preventing influence of animal-derived serum ingredients on cell culture. The medium can be used for studying the differentiation and proliferation adjusting mechanism of the umbilical cord mesenchymal stem cells.
Description
Technical field
The present invention relates to technical field of cell culture, specifically a kind of umbilical cord mesenchymal stem cells substratum of serum-free.
Background technology
Umbilical cord mesenchymal stem cells refers to a kind of multipotential stem cell be present in neonatal umbilical cord tissue, cell surface strongly expressed specific marker CD90, CD73, CD105, and do not express mark CD45, CD14, CD11, CD34, CD19 of hematopoiesis system or interior skin system cell, low expression or do not express major histocompatibility antigen HLA-DR.Research display, umbilical cord mesenchymal stem cells has higher differentiation potential, can carry out differentiation as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium and cardiac muscle etc. to multiple directions.Compared with mesenchymal stem cells MSCs, umbilical cord mesenchymal stem cells content and multiplication capacity are all better than the latter, and immunogenicity low, draw materials conveniently, without advantages such as ethics disputes, therefore there is in mescenchymal stem cell higher clinical value.
In practical clinical, be the necessary factor ensureing umbilical cord mesenchymal stem cells treatment without exogenous pollution and cell quantity.At present, the culture system of umbilical cord mesenchymal stem cells is mostly containing animal serum (as foetal calf serum), on the one hand due to the existence of albumen not clear in serum, to signal transduction and the cell surface marker formation interference of cell, the stem cell of adding serum free culture system is adopted to carry out transplanting in body on the other hand, the cross infection of pathogenic agent may be caused, therefore need the serum free medium developing specific chemical components.At present, when the serum-free stem cell media that market exists is cultivated for cultivating umbilical cord mesenchymal stem cells, a part is due to the deficiency of serum free medium composition aspect, the speed of growth is slow, the aging feature of easy appearance affects a large amount of amplifications of cell, with the addition of the specific factors such as expensive antibody in part substratum, add umbilical cord mesenchymal stem cells amplification cost.Above drawback hinders the clinical treatment application of umbilical cord mesenchymal stem cells to a certain extent.
Summary of the invention
Technical assignment of the present invention solves the deficiencies in the prior art, provides a kind of umbilical cord mesenchymal stem cells substratum of serum-free, overcomes serum exogenous pollution defect and cell amplification quantity and contradictory problems between reducing costs.
Technical scheme of the present invention realizes in the following manner, the experimental technique of a kind of umbilical chord mesenchymal cells Secondary Culture and amplification in the umbilical cord mesenchymal stem cells substratum of serum-free, and the step of this experimental technique is:
(1) first prepare the umbilical cord mesenchymal stem cells substratum of serum-free, the umbilical cord mesenchymal stem cells substratum of this serum-free comprises basic medium and adds composition; Wherein, basic medium is DMEM substratum, add composition and content as follows:
Basic Fibroblast Growth Factor hFGF:1-20ng/mL;
Urogastron hEGF:1-20 μ g/mL;
Regular Insulin hI:1-20 μ g/mL;
Leukaemia inhibitory factor hLIF:0.1-5ng/mL;
L-glutaminate: 1-10mM;
2 mercapto ethanol: 50-200 μM;
Sodium Selenite: 20-50nM;
Transferrins,iron complexes: 10-30 μ g/mL;
Human albumin: 10-100mg/mL;
Fibronectin: 50-200 μ g/mL;
Astragalus polysaccharides APS:50-200 μ g/mL.
(2) experimental procedure of umbilical chord mesenchymal cells Secondary Culture and amplification in above-mentioned substratum is:
A. and the umbilical cord mesenchymal stem cells cryopreservation tube of Liquid nitrogen storage separating obtained through conventional method is taken out from liquid nitrogen, drop in 37 DEG C of water-baths immediately, gentle agitation, i.e. solubilized in 2min, the umbilical cord mesenchymal stem cells substratum of the above-mentioned serum-free of preheating simultaneously;
B. the umbilical cord mesenchymal stem cells after thawing through 37 DEG C is drawn onto in the centrifuge tube of the 15mL that 9mL substratum is housed, washes 2 cryopreservation tubes with nutrient solution simultaneously, be added to together in centrifuge tube and blow even, avoid producing foam, then centrifugal 5 minutes of the rotating speed of 250 times of gravity;
C. abandon supernatant liquor, add 2mL preheating respectively
?above-mentioned substratum, blows even gently, ensures that cell suspends completely; Resuspended cell is inoculated in the culturing bottle of T25, adds appropriate nutrient solution, then cell is shaken evenly, ensure that all cells can distribute uniformly;
D. mark cell category and date, kinds of culture medium etc., put into 37 DEG C, 5%CO
2in incubator;
E. change substratum after cell attachment, ensure to remove dead cell;
Cell fraction of coverage in culture dish is passaged in Tissue Culture Plate in the ratio of 1:3 when reaching 80% ~ 90%, and the growth medium used that goes down to posterity is substratum of the present invention; Basis of microscopic observation umbilical cord mesenchymal stem cells upgrowth situation; To take pictures record;
When f. reaching 2nd generation, mtt assay detects cytoactive and draws cell growth curve, concrete operations: suspended by the 2nd generation umbilical cord mesenchymal stem cells enzymolysis growing to 80% degrees of fusion, adjustment cell concn to 1 × 10
4individual/mL, adds 24 orifice plates, every hole 800 μ L, be placed in 37 DEG C, saturated humidity, 5% CO
2cultivate in incubator, a 24 orifice plates are taken out every 6 hours, it is after the MTT solution of 5g/L that every hole adds 80 μ L concentration, 4h is placed at 37 DEG C, add the dimethyl sulfoxide (DMSO) DMSO of 600 μ L in every hole, measure each hole absorption photometric OD value by microplate reader at 550nm place, represent the relative populations of cell with OD value, draw growth curve, record result;
(3) testing sequence of the Surface marker analysis of culturing cell is:
Get the cell in P2 generation, remove nutrient solution, PBS washs 2 times, with the 0.05% mass concentration trypsin solution digestion of 1:1, the centrifugal 5min of 1000r/min, washs 1 time with PBS, adjustment cell concn, makes the single cell suspension that concentration is 106/mL, adds 10 μ L antibody, 30min is hatched at 4 DEG C, 1 time is washed, the centrifugal 5min of 1000r/min, with the PBS re-suspended cell of 500 μ L with PBS, then flow cytometer detects, and contrasts.
The beneficial effect that the present invention is compared with prior art produced is:
The umbilical cord mesenchymal stem cells substratum of a kind of serum-free of the present invention overcomes serum exogenous pollution defect and cell amplification quantity and contradictory problems between reducing costs.
The umbilical cord mesenchymal stem cells substratum of a kind of serum-free of the present invention, not containing serum, can avoid animal source serum composition on the impact of cell cultures; Composition is clear and definite, can be used for the differentiation and proliferation regulation mechanism studying umbilical cord mesenchymal stem cells; With the addition of the propagation that vegetable polysaccharides astragalus polysaccharides promotes umbilical cord mesenchymal stem cells in substratum, substitute the distinct antibodies factor, reduce cost.
The umbilical cord mesenchymal stem cells be incubated in the umbilical cord mesenchymal stem cells substratum of serum-free of the present invention keeps good adherent characteristic, and umbilical cord mesenchymal stem cells all presents the one-tenth threadiness cellular form of fusiformis.
Umbilical cord mesenchymal stem cells in growth medium of the present invention can grow to plateau before 54 hours.But the umbilical cord mesenchymal stem cells in contrast growth medium can only can grow to plateau after 66 hours.This shows, growth medium of the present invention can accelerate the speed of growth of umbilical cord mesenchymal stem cells effectively.
Serum free medium of the present invention and the display of control group culture medium culturing MSC fluidic cell phenotype comparing result, serum free medium provided by the invention and control group substratum MSC positive expression CD73, CD90, CD105, negative expression CD14, CD19, CD34 and CD45, do not have significant difference.
Accompanying drawing explanation
fig. 1a and
fig. 1b is the form of the umbilical cord mesenchymal stem cells that different dry cell culture medium is cultivated
figuresheet:
fig. 1a is the form of the umbilical cord mesenchymal stem cells of the umbilical cord mesenchymal stem cells culture medium culturing of serum-free of the present invention
figuresheet;
fig. 1b is the form of the umbilical cord mesenchymal stem cells that common stem cell media is cultivated
figuresheet.
fig. 2the growth curve of the umbilical cord mesenchymal stem cells grown in different growth mediums
figure:
fig. 2in 1 curve be the growth curve of the umbilical cord mesenchymal stem cells culture medium culturing of serum-free of the present invention
figure;
fig. 2in 2 curves be the growth curve of umbilical cord mesenchymal stem cells that common stem cell media is cultivated
figure;
fig. 2in, X-coordinate represents growth time (unit for hour), and ordinate zou represents 550nm place mensuration each hole absorption photometric value (OD value).
Embodiment
Below the umbilical cord mesenchymal stem cells substratum of a kind of serum-free of the present invention is described in detail below.
The umbilical cord mesenchymal stem cells substratum of a kind of serum-free of the present invention, and utilize this substratum to make the experimental technique of umbilical chord mesenchymal cells Secondary Culture and amplification ability, its experiment implemented is:
(1) combination experiment of cytokine in serum free medium:
Selected somatomedin: recombinant human fibroblast growth factor 2 (hFGF), biosynthetic human insulin (hI), rhEGF (hEGF), people's recombinant leukemia inhibitor factor (hLIF) and astragalus polysaccharides (APS) concentrated solution, through sterile filtration, refrigeration or cold storage for subsequent use.
For different somatomedins design different concentration (
table 1) to add in nutrient solution and to find the optimum effect concentration of this somatomedin according to growth curve.Through the screening experiment of over-richness the different somatomedins of optimum concn are carried out combine (
table 2), the respective impact on stem cell growth situation of investigation (
table 3), obtain optimum growh combinations of factors nutrient solution.
table 1the concentration of various somatomedin
table 2cytokine and concentration combination table
table 3the contrast of growth and proliferation of cell in the substratum system of various combination
table 4range analysis result
table 4that a day Honda mouth orthogonal test is obtained a result.
According to
table 4interpretation of result: cell harvesting amount and required cost viewpoint after the identical number of days of Synthetical cultivation, each somatomedin optimum effect concentration is finally defined as:
The optimum concn of hFGF is 10ng/ml,
The optimum concn of hEGF is 10 μ g/ml,
The optimum concn of hI is 10 μ g/ml,
The optimum effect concentration of hLIF is 1ng/ml,
The optimum effect concentration of APS is 0.1mg/ml.
(2) umbilical chord mesenchymal cells in the medium Secondary Culture and amplification ability comparative analysis
1. and the umbilical cord mesenchymal stem cells cryopreservation tube of Liquid nitrogen storage separating obtained through conventional method is taken out from liquid nitrogen, drop in 37 DEG C of water-baths immediately, gentle agitation (dissolving in 2min), the above-mentioned substratum of preheating simultaneously.
2. the umbilical cord mesenchymal stem cells after thawing through 37 DEG C is drawn onto in the centrifuge tube of the 15mL that 9mL substratum is housed, washes 2 cryopreservation tubes with nutrient solution simultaneously, be added to together in centrifuge tube and blow even, avoid producing foam.Then centrifugal 5 minutes of 250G rotating speed.
3. abandon supernatant liquor, add the above-mentioned substratum of 2mL preheating respectively, blow even gently, ensure that cell suspends completely; Resuspended cell is inoculated in the culturing bottle of T25, adds appropriate nutrient solution, then cell is shaken evenly, ensure that all cells can distribute uniformly.
4. mark cell category and date, kinds of culture medium etc., put into 37 DEG C, 5%CO
2in incubator.
5. change substratum after cell attachment, ensure to remove dead cell.Ratio in 1: 3 when cell fraction of coverage in culture dish reaches 80% ~ 90% is passaged in Tissue Culture Plate, and the growth medium used that goes down to posterity is substratum of the present invention and common stem cell media.Basis of microscopic observation umbilical cord mesenchymal stem cells upgrowth situation.
fig. 1shown in A, the umbilical cord mesenchymal stem cells be incubated in the umbilical cord mesenchymal stem cells substratum of serum-free of the present invention keeps good adherent characteristic, and umbilical cord mesenchymal stem cells all presents the one-tenth threadiness cellular form of fusiformis.
6. when reaching 2nd generation, mtt assay detects cytoactive and draws cell growth curve, concrete operations: suspended by the 2nd generation umbilical cord mesenchymal stem cells enzymolysis growing to 80% degrees of fusion, adjustment cell concn to 1 × 10
4individual/mL, adds 24 orifice plates, every hole 800 μ L, be placed in 37 DEG C, saturated humidity, 5% CO
2cultivate in incubator, a 24 orifice plates are taken out every 6 hours, after every hole adds the MTT solution (5g/L) of 80 μ L, 4h is placed at 37 DEG C, add the dimethyl sulfoxide (DMSO) (DMSO) of 600 μ L in every hole, measure each hole absorption photometric value (OD value) by microplate reader at 550nm place, represent the relative populations of cell with OD value, draw growth curve, result
as Fig. 2shown in, the umbilical cord mesenchymal stem cells in growth medium of the present invention can grow to plateau before 54 hours.But the umbilical cord mesenchymal stem cells in contrast growth medium can only can grow to plateau after 66 hours.This shows, growth medium of the present invention can accelerate the speed of growth of umbilical cord mesenchymal stem cells effectively.
(3) Surface marker analysis of culturing cell
Get the cell in P2 generation, remove nutrient solution, PBS washs 2 times, and with the 0.05% trypsin solution digestion of 1: 1, the centrifugal 5min of 1000r/min, washs 1 time with PBS, and adjustment cell concn, making concentration is 10
6the single cell suspension of/ml, adds 10 μ L antibody, hatches 30min, wash 1 time with PBS at 4 DEG C, the centrifugal 5min of 1000r/min, and with the PBS re-suspended cell of 500 μ L, then flow cytometer detects, and contrasts.The results are shown in
table 4.Serum free medium of the present invention and the display of control group culture medium culturing MSC fluidic cell phenotype comparing result, serum free medium provided by the invention and control group substratum MSC positive expression CD73, CD90, CD105, negative expression CD14, CD19, CD34 and CD45, do not have significant difference.
table 5p2 is for cell surface molecule marker expression per-cent
DMEM described in present method is a kind of substratum containing each seed amino acid and glucose, is develop on the basis of MEM substratum.Compare with MEM and add various Ingredient Amount, be divided into again high glycoform (lower than 4500mg/L) and low-sugar type (lower than 1000mg/L) simultaneously.High glycoform is conducive to cell and berths in a position growth, is suitable for growing comparatively fast, adheres to more difficult tumour cell etc.
DMEM described in present method is the abbreviation of dulbecco'smodifiedeaglemedium, so its feature mainly comprises following: (1) aminoacids content is 2 times of Yi Geer substratum, and containing non-essential amino acid, as glycine etc.; (2) vitamin contents is 4 times of Yi Geer substratum; (3) containing the important substance in glycolytic pathway---pyruvic acid; (4) iron ion containing trace.
This DMEM substratum is widely used in production of vaccine and the various first cell cultures for virus host cells and single cell and cultivates; As the cultivation of the clones such as A9,3T6, BALB/3T3, COS-1, COS-3, COS-7, L6, WEHI-3b.
DMEM substratum described in present method can adopt the DMEM substratum of following compositions.
DMEM cell culture medium composition:
| Sequence number | Compound title | Content (mg/L) | Sequence number | Compound title | Content (mg/L) |
| 1 | Calcium Chloride Powder Anhydrous 2H 2O | 265.00 | 18 | Serine | 42.00 |
| 2 | Iron nitrate 9H 2O | 0.10 | 19 | L-threonine | 95.00 |
| 3 | Repone K | 400.00 | 20 | L-Trp | 16.00 |
| 4 | Anhydrous magnesium sulfate | 97.67 | 21 | TYR | 72.00 |
| 5 | Sodium-chlor | 6400.00 | 22 | Valine | 94.00 |
| 6 | AMSP | 109.00 | 23 | D-VB5 calcium | 4.00 |
| 7 | Succinic acid | 75.00 | 24 | Choline tartrate | 7.20 |
| 8 | Soduxin | 100.00 | 25 | Folic acid | 4.00 |
| 9 | L-arginine hydrochloride | 84.00 | 26 | Inositol | 7.20 |
| 10 | L-hydrochloric acid Gelucystine | 63.00 | 27 | Niacinamide | 4.00 |
| 11 | Glycine | 30.00 | 28 | Riboflavin | 0.40 |
| 12 | L-histidine monohydrochloride | 42.00 | 29 | Thiamine hydrochloride | 4.00 |
| 13 | ILE | 105.00 | 30 | Pyridoxine hydrochloride | 4.00 |
| 14 | L-Leu | 105.00 | 31 | Glucose | 1000.00 |
| 15 | LYS | 146.00 | 32 | Sodium.alpha.-ketopropionate | 110.00 |
| 16 | METHIONINE | 30.00 | 33 | Phenol red | 9.03 |
| 17 | L-Phe | 66.00 |
Cell MTT described in present method tests:
MTT full name is 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide, Chinese chemistry 3-(4 by name, 5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt, trade(brand)name: tetrazolium bromide.It is a kind of dyestuff of yellow color.
MTT mainly contains two purposes:
1. medicine (also comprising other processing modes as radiation exposure) the Cytotoxic mensuration to vitro culture;
2. cell proliferation and cytoactive detection.
MTT Cleaning Principle is that the succinodehydrogenase in viable cell plastosome can make exogenous MTT be reduced to water-insoluble bluish voilet Jie Jing Jia Za (Z ā) (Formazan) and be deposited in cell, and dead cell is without this function.Jia Za (Z ā) in dimethyl sulfoxide (DMSO) (DMSO) energy dissolved cell, its absorbance value is measured at 490nm wavelength place (English illustrate write be 570nm) by microplate reader, within the scope of certain cell count, the amount that MTT crystallization is formed is directly proportional to cell count.According to the absorbance recorded (OD value), judge viable cell quantity, OD value is larger, cytoactive stronger (if survey drug toxicity, then representing that drug toxicity is less).
The umbilical cord mesenchymal stem cells substratum of a kind of serum-free of the present invention, not containing serum, can avoid animal source serum composition on the impact of cell cultures; Composition is clear and definite, can be used for the differentiation and proliferation regulation mechanism studying umbilical cord mesenchymal stem cells; With the addition of the propagation that vegetable polysaccharides astragalus polysaccharides promotes umbilical cord mesenchymal stem cells in substratum, substitute the distinct antibodies factor, reduce cost.
Serum free medium of the present invention and the display of control group culture medium culturing MSC fluidic cell phenotype comparing result, serum free medium provided by the invention and control group substratum MSC positive expression CD73, CD90, CD105, negative expression CD14, CD19, CD34 and CD45, do not have significant difference.
Claims (4)
1. a umbilical cord mesenchymal stem cells substratum for serum-free, is characterized in that comprising basic medium and adding composition; Wherein, basic medium is DMEM substratum, add composition and content as follows:
Basic Fibroblast Growth Factor hFGF:1-20ng/mL;
Urogastron hEGF:1-20 μ g/mL;
Regular Insulin hI:1-20 μ g/mL;
Leukaemia inhibitory factor hLIF:0.1-5ng/mL;
L-glutaminate: 1-10mM;
2 mercapto ethanol: 50-200 μM;
Sodium Selenite: 20-50nM;
Transferrins,iron complexes: 10-30 μ g/mL;
Human albumin: 10-100mg/mL;
Fibronectin: 50-200 μ g/mL;
Astragalus polysaccharides APS:50-200 μ g/mL.
2. a umbilical cord mesenchymal stem cells substratum for serum-free as claimed in claim 1, add composition and content as follows:
Basic Fibroblast Growth Factor hFGF:5-15ng/mL, preferred 10ng/mL;
Urogastron hEGF:5-15 μ g/mL, preferably 10 μ g/mL;
Regular Insulin hI:5-18 μ g/mL, preferably 10 μ g/mL;
Leukaemia inhibitory factor hLIF:0.5-2.5ng/mL, preferential 1ng/mL;
L-glutaminate: 5-8mM, preferred 5mM;
2 mercapto ethanol: 80-150 μM, preferably 100 μMs;
Sodium Selenite: 25-40nM, preferred 30nM;
Transferrins,iron complexes: 15-25 μ g/mL, preferably 25 μ g/mL;
Human albumin: 30-80mg/mL, preferred 50mg/mL;
Fibronectin: 80-150 μ g/mL, preferably 100 μ g/mL;
Astragalus polysaccharides APS:80-150 μ g/mL, preferably 100 μ g/mL.
3. the Secondary Culture of umbilical chord mesenchymal cells and a method for amplification, is characterized in that the step of this experimental technique is:
(1) the umbilical cord mesenchymal stem cells substratum as the serum-free of any one of claim 1 ~ 2 is first prepared;
(2) Secondary Culture and amplification step:
A. and the umbilical cord mesenchymal stem cells cryopreservation tube of Liquid nitrogen storage separating obtained through conventional method is taken out from liquid nitrogen, drop in 37 DEG C of water-baths immediately, gentle agitation, i.e. solubilized in 2min, the umbilical cord mesenchymal stem cells substratum of the above-mentioned serum-free of preheating simultaneously;
B. the umbilical cord mesenchymal stem cells after thawing through 37 DEG C is drawn onto in the centrifuge tube of the 15mL that 9mL substratum is housed, washes 2 cryopreservation tubes with nutrient solution simultaneously, be added to together in centrifuge tube and blow even, avoid producing foam, then centrifugal 5 minutes of the rotating speed of 250 times of gravity;
C. abandon supernatant liquor, add respectively 2mL preheating above-mentioned substratum, blow even gently, ensure cell suspend completely; Resuspended cell is inoculated in the culturing bottle of T25, adds appropriate nutrient solution, then cell is shaken evenly, ensure that all cells can distribute uniformly;
D. mark cell category and date, kinds of culture medium etc., put into 37 DEG C, 5%CO
2in incubator;
E. change substratum after cell attachment, ensure to remove dead cell;
Cell fraction of coverage in culture dish is passaged in Tissue Culture Plate in the ratio of 1:3 when reaching 80% ~ 90%, and the growth medium used that goes down to posterity is substratum of the present invention; Basis of microscopic observation umbilical cord mesenchymal stem cells upgrowth situation; To take pictures record;
When f. reaching 2nd generation, mtt assay detects cytoactive and draws cell growth curve, concrete operations: suspended by the 2nd generation umbilical cord mesenchymal stem cells enzymolysis growing to 80% degrees of fusion, adjustment cell concn to 1 × 10
4individual/mL, adds 24 orifice plates, every hole 800 μ L, be placed in 37 DEG C, saturated humidity, 5% CO
2cultivate in incubator, a 24 orifice plates are taken out every 6 hours, it is after the MTT solution of 5g/L that every hole adds 80 μ L concentration, 4h is placed at 37 DEG C, add the dimethyl sulfoxide (DMSO) DMSO of 600 μ L in every hole, measure each hole absorption photometric OD value by microplate reader at 550nm place, represent the relative populations of cell with OD value, draw growth curve, record result.
4. a kind of Secondary Culture of umbilical chord mesenchymal cells as claimed in claim 3 and the method for amplification, it is characterized in that the method comprises the testing sequence of the Surface marker analysis of step (three) culturing cell further: the cell getting P2 generation, remove nutrient solution, PBS washs 2 times, with the 0.05% mass concentration trypsin solution digestion of 1:1, the centrifugal 5min of 1000r/min, 1 time is washed with PBS, adjustment cell concn, make the single cell suspension that concentration is 106/mL, add 10 μ L antibody, 30min is hatched at 4 DEG C, 1 time is washed with PBS, the centrifugal 5min of 1000r/min, with the PBS re-suspended cell of 500 μ L, then flow cytometer detects, contrast.
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